The goal of this project is to obtain residue-specific characterization of the receptor binding sites and antigenic signatures on the envelope proteins of Venezuelan Equine Encephalitis virus (VEEV) and eventually for all alphaviruses. The Center for Disease Control and Prevention has classified VEEV as a bioterrorism agent, as it can be grown to high titers, and disseminated by aerosol. We will test the hypothesis that a comprehensive bioinformatics approach, combined with assays for cell binding, infection, and neutralization with monoclonal antibodies, will identify areas of the glycoproteins that determine antigenicity, fusion, and receptor interactions with host cells. We will establish a 3D-database of interacting regions of known complexes and new tools to locate areas with similar physical chemical properties in other proteins. The resulting trained neural networks or support vector machines will be used to identify areas of the envelope glycoproteins E1 and E2 of VEEV that are most likely to interact. As the 3D structures of E1 and E2 of VEEV have not been experimentally determined, we will model them on existing templates. We will map mutations of El and E2 that correlate with virulence on the models and analyze the structures of the surrounding areas. Mutants in selected areas will be assayed for their role in viral entry and fusion, using murine leukemia virus (MLV) pseudotypes that express the VEEV envelope proteins. This novel expression system permits study of the interacting sites of VEEV without the risks of dealing with infectious virus. Once the interacting areas of VEEV have been determined, our computational analysis will be extended to other pathogenic members of the alphavirus family. The resulting models can be used to design peptides mimicking the receptor recognition motifs, that may be the basis for the design of vaccines or inhibitors against alphaviruses.